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Foxp2 controls synaptic wiring of corticostriatal circuits and vocal communication by opposing Mef2c

Abstract

Cortico-basal ganglia circuits are critical for speech and language and are implicated in autism spectrum disorder, in which language function can be severely affected. We demonstrate that in the mouse striatum, the gene Foxp2 negatively interacts with the synapse suppressor gene Mef2c. We present causal evidence that Mef2c inhibition by Foxp2 in neonatal mouse striatum controls synaptogenesis of corticostriatal inputs and vocalization in neonates. Mef2c suppresses corticostriatal synapse formation and striatal spinogenesis, but can itself be repressed by Foxp2 through direct DNA binding. Foxp2 deletion de-represses Mef2c, and both intrastriatal and global decrease of Mef2c rescue vocalization and striatal spinogenesis defects of Foxp2-deletion mutants. These findings suggest that Foxp2–Mef2C signaling is critical to corticostriatal circuit formation. If found in humans, such signaling defects could contribute to a range of neurologic and neuropsychiatric disorders.

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Figure 1: Dissociation of Foxp2 and Mef2C in SPNs is initiated in VGluT1+ striosomes in neonatal striatum.
Figure 2: Progressive dissociation of Foxp2 and Mef2C in SPNs during postnatal development.
Figure 3: Synaptic proteins and dendritic spines of SPNs are oppositely regulated by Foxp2 and Mef2c.
Figure 4: Foxp2 suppresses Mef2c expression in SPNs.
Figure 5: Mef2c is a direct target gene of Foxp2.
Figure 6: Inactivation of one allele of Mef2c rescues USV, dendritic spines and synaptic protein changes in P8 Foxp2+/− mice.
Figure 7: Reduction of USVs in Nestin-cre;Mef2c knockout mice at P8.

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References

  1. Abrahams, B.S. & Geschwind, D.H. Advances in autism genetics: on the threshold of a new neurobiology. Nat. Rev. Genet. 9, 341–355 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Mody, M. & Belliveau, J.W. Speech and language impairments in autism: insights from behavior and neuroimaging. N. Am. J. Med. Sci. (Boston) 5, 157–161 (2013).

    Google Scholar 

  3. Hollander, E. et al. Striatal volume on magnetic resonance imaging and repetitive behaviors in autism. Biol. Psychiatry 58, 226–232 (2005).

    PubMed  Google Scholar 

  4. Di Martino, A. et al. Aberrant striatal functional connectivity in children with autism. Biol. Psychiatry 69, 847–856 (2011).

    PubMed  Google Scholar 

  5. Watkins, K.E. et al. MRI analysis of an inherited speech and language disorder: structural brain abnormalities. Brain 125, 465–478 (2002).

    CAS  PubMed  Google Scholar 

  6. Vargha-Khadem, F. et al. Neural basis of an inherited speech and language disorder. Proc. Natl. Acad. Sci. USA 95, 12695–12700 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Groszer, M. et al. Impaired synaptic plasticity and motor learning in mice with a point mutation implicated in human speech deficits. Curr. Biol. 18, 354–362 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Enard, W. et al. A humanized version of Foxp2 affects cortico-basal ganglia circuits in mice. Cell 137, 961–971 (2009).

    CAS  PubMed  Google Scholar 

  9. Graham, S.A. & Fisher, S.E. Decoding the genetics of speech and language. Curr. Opin. Neurobiol. 23, 43–51 (2013).

    CAS  PubMed  Google Scholar 

  10. Mukamel, Z. et al. Regulation of MET by FOXP2, genes implicated in higher cognitive dysfunction and autism risk. J. Neurosci. 31, 11437–11442 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Vernes, S.C. et al. A functional genetic link between distinct developmental language disorders. N. Engl. J. Med. 359, 2337–2345 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Roll, P. et al. Molecular networks implicated in speech-related disorders: FOXP2 regulates the SRPX2/uPAR complex. Hum. Mol. Genet. 19, 4848–4860 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Zoghbi, H.Y. & Bear, M.F. Synaptic dysfunction in neurodevelopmental disorders associated with autism and intellectual disabilities. Cold Spring Harb. Perspect. Biol. 4, a009886 (2012).

    PubMed  PubMed Central  Google Scholar 

  14. Ebert, D.H. & Greenberg, M.E. Activity-dependent neuronal signalling and autism spectrum disorder. Nature 493, 327–337 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Novara, F. et al. Refining the phenotype associated with MEF2C haploinsufficiency. Clin. Genet. 78, 471–477 (2010).

    CAS  PubMed  Google Scholar 

  16. Rauch, A. et al. MEF2C mutations are a frequent cause of Rett- or Angelman syndrome like neurodevelopmental disorders. 12th International Congress of Human Genetics/61st Annual Meeting of the American Society of Human Genetics. Program Number: 1026T, http://www.ashg.org/2011meeting/pdf/ICHG%20Poster%20Abstracts.pdf (2011).

  17. Neale, B.M. et al. Patterns and rates of exonic de novo mutations in autism spectrum disorders. Nature 485, 242–245 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Spiteri, E. et al. Identification of the transcriptional targets of FOXP2, a gene linked to speech and language, in developing human brain. Am. J. Hum. Genet. 81, 1144–1157 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Le Meur, N. et al. MEF2C haploinsufficiency caused by either microdeletion of the 5q14.3 region or mutation is responsible for severe mental retardation with stereotypic movements, epilepsy and/or cerebral malformations. J. Med. Genet. 47, 22–29 (2010).

    CAS  PubMed  Google Scholar 

  20. Flavell, S.W. et al. Activity-dependent regulation of MEF2 transcription factors suppresses excitatory synapse number. Science 311, 1008–1012 (2006).

    CAS  PubMed  Google Scholar 

  21. Barbosa, A.C. et al. MEF2C, a transcription factor that facilitates learning and memory by negative regulation of synapse numbers and function. Proc. Natl. Acad. Sci. USA 105, 9391–9396 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Tsai, N.P. et al. Multiple autism-linked genes mediate synapse elimination via proteasomal degradation of a synaptic scaffold PSD-95. Cell 151, 1581–1594 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Tian, X., Kai, L., Hockberger, P.E., Wokosin, D.L. & Surmeier, D.J. MEF-2 regulates activity-dependent spine loss in striatopallidal medium spiny neurons. Mol. Cell. Neurosci. 44, 94–108 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Nisenbaum, L.K., Webster, S.M., Chang, S.L., McQueeney, K.D. & LoTurco, J.J. Early patterning of prelimbic cortical axons to the striatal patch compartment in the neonatal mouse. Dev. Neurosci. 20, 113–124 (1998).

    CAS  PubMed  Google Scholar 

  25. Somogyi, P., Bolam, J.P. & Smith, A.D. Monosynaptic cortical input and local axon collaterals of identified striatonigral neurons. A light and electron microscopic study using the Golgi-peroxidase transport-degeneration procedure. J. Comp. Neurol. 195, 567–584 (1981).

    CAS  PubMed  Google Scholar 

  26. Sheth, A.N., McKee, M.L. & Bhide, P.G. The sequence of formation and development of corticostriate connections in mice. Dev. Neurosci. 20, 98–112 (1998).

    CAS  PubMed  Google Scholar 

  27. Fremeau, R.T. Jr., Voglmaier, S., Seal, R.P. & Edwards, R.H. VGLUTs define subsets of excitatory neurons and suggest novel roles for glutamate. Trends Neurosci. 27, 98–103 (2004).

    CAS  PubMed  Google Scholar 

  28. Harris, K.M., Jensen, F.E. & Tsao, B. Three-dimensional structure of dendritic spines and synapses in rat hippocampus (CA1) at postnatal day 15 and adult ages: implications for the maturation of synaptic physiology and long-term potentiation. J. Neurosci. 12, 2685–2705 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Li, S., Weidenfeld, J. & Morrisey, E.E. Transcriptional and DNA binding activity of the Foxp1/2/4 family is modulated by heterotypic and homotypic protein interactions. Mol. Cell. Biol. 24, 809–822 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Shu, W. et al. Altered ultrasonic vocalization in mice with a disruption in the Foxp2 gene. Proc. Natl. Acad. Sci. USA 102, 9643–9648 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Li, H. et al. Transcription factor MEF2C influences neural stem/progenitor cell differentiation and maturation in vivo. Proc. Natl. Acad. Sci. USA 105, 9397–9402 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Pulipparacharuvil, S. et al. Cocaine regulates MEF2 to control synaptic and behavioral plasticity. Neuron 59, 621–633 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Akhtar, M.W. et al. In vivo analysis of MEF2 transcription factors in synapse regulation and neuronal survival. PLoS One 7, e34863 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Adachi, M., Lin, P.Y., Pranav, H. & Monteggia, L.M. Postnatal loss of Mef2c results in dissociation of effects on synapse number and learning and memory. Biol. Psychiatry 80, 140–148 (2016).

    CAS  PubMed  Google Scholar 

  35. Sia, G.M., Clem, R.L. & Huganir, R.L. The human language-associated gene SRPX2 regulates synapse formation and vocalization in mice. Science 342, 987–991 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Schulz, S.B., Haesler, S., Scharff, C. & Rochefort, C. Knockdown of FoxP2 alters spine density in Area X of the zebra finch. Genes Brain Behav. 9, 732–740 (2010).

    CAS  PubMed  Google Scholar 

  37. Haesler, S. et al. Incomplete and inaccurate vocal imitation after knockdown of FoxP2 in songbird basal ganglia nucleus Area X. PLoS Biol. 5, e321 (2007).

    PubMed  PubMed Central  Google Scholar 

  38. Chahrour, M. et al. MeCP2, a key contributor to neurological disease, activates and represses transcription. Science 320, 1224–1229 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Morrow, E.M. et al. Identifying autism loci and genes by tracing recent shared ancestry. Science 321, 218–223 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Crittenden, J.R. & Graybiel, A.M. Basal ganglia disorders associated with imbalances in the striatal striosome and matrix compartments. Front. Neuroanat. 5, 59 (2011).

    PubMed  PubMed Central  Google Scholar 

  41. Lipton, S.A. et al. Autistic phenotype from MEF2C knockout cells. Science 323, 208 (2009).

    CAS  PubMed  Google Scholar 

  42. Tronche, F. et al. Disruption of the glucocorticoid receptor gene in the nervous system results in reduced anxiety. Nat. Genet. 23, 99–103 (1999).

    CAS  PubMed  Google Scholar 

  43. Stenman, J., Toresson, H. & Campbell, K. Identification of two distinct progenitor populations in the lateral ganglionic eminence: implications for striatal and olfactory bulb neurogenesis. J. Neurosci. 23, 167–174 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Lu, K.M., Evans, S.M., Hirano, S. & Liu, F.C. Dual role for Islet-1 in promoting striatonigral and repressing striatopallidal genetic programs to specify striatonigral cell identity. Proc. Natl. Acad. Sci. USA 111, E168–E177 (2014).

    CAS  PubMed  Google Scholar 

  45. Ouimet, C.C., Miller, P.E., Hemmings, H.C. Jr., Walaas, S.I. & Greengard, P. DARPP-32, a dopamine- and adenosine 3′:5′-monophosphate-regulated phosphoprotein enriched in dopamine-innervated brain regions. III. Immunocytochemical localization. J. Neurosci 4, 111–124 (1984).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Chang, S.L. et al. Ectopic expression of Nolz-1 in neural progenitors promotes cell cycle exit/premature neuronal differentiation accompanying with abnormal apoptosis in the developing mouse telencephalon. PLoS One 8, e74975 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Kaoru, T., et al. Molecular characterization of the intercalated cell masses of the amygdala: implications for the relationship with the striatum. Neuroscience 166, 220–230 (2010).

    CAS  PubMed  Google Scholar 

  48. Pawlak, V. & Kerr, J.N. Dopamine receptor activation is required for corticostriatal spike-timing-dependent plasticity. J. Neurosci. 28, 2435–2446 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank P. Arrollta of Harvard University, K. Campbell of Cincinnati Children's Hospital Medical Center, H.C. Hemmings of Rockefeller University, O. Marin of King's College London and E. Olson of University of Texas Southwestern Medical Center for providing transgenic mice and reagents; M. Bear, M. Sur, M.-M. Poo and D. Homma for discussion; and Y. Kubota for help with illustrations and manuscript preparation. This work was supported by NIH/NICHD grant R37 HD028341 (A.M.G.), the Nancy Lurie Marks Family Foundation (A.M.G.), the Simons Center for the Social Brain at MIT (A.M.G.), the Paul G. Allen Family Foundation (S.P.), National Science Council grants NSC97-2321-B-010-006, NSC98-2321-B-010-002, NSC99-2321-B-010-002, NSC100-2321-B-010-002, NSC101-2321-B-010-021, NSC102-2321-B-010-018 and NSC102-2911-I-010-506 (F.-C.L.), Ministry of Science and Technology grants MOST103-2321-B-010-009 and MOST104-2321-B-010-022 (F.-C.L.), National Health Research Institutes grants NHRI-EX104-10429NI and NHRI-EX105-10429NI (F.-C.L.) and by an Aiming for Top University grant from the Ministry of Education, Taiwan (F.-C.L.).

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Authors and Affiliations

Authors

Contributions

F.-C.L. conceived and supervised the project. Y.-C. Chen, H.-Y.K., H.T., S.-Y.C., K.-M.L., C.-T.C., U.B., W.H., W.E., S.P., A.M.G. and F.-C.L. designed the experiments. Y.-C. Chen, H.-Y.K., U.B., W.H., S.-Y.C., H.-Y.Y., G.-M.C., Y.-H.L. and S.-J.C. performed experiments on Foxp2 and Mef2c mutant mice; K.-M.L., J.-R.L. and Y.-C. Chou performed Foxp2 and Mef2C overexpression experiments; H.T., Y.-C. Chen and S.-Y.C. characterized Foxp2 binding sites; and W.E. and S.P. provided Foxp2 mutant mice and interpretation of data and discussion. Y.-C. Chen., H.-Y.K., S.-Y.C., K.-M.L., H.T., U.B., W.H., W.E., S.P., A.M.G. and F.-C.L. analyzed the data. A.M.G. and F.-C.L. wrote the paper with input from all authors.

Corresponding authors

Correspondence to Ann M Graybiel or Fu-Chin Liu.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Specificity of Foxp2 and Mef2C antibodies and postnatal development of Foxp2 and Mef2C in neocortex.

(a-f) Foxp2 and Mef2C immunoreactive signals are barely detectable in Foxp2−/− knockout striatum (a-c; Student's t test, t(4) = 56.963, P = 0.000001) and Nestin- cre;Mef2 cfl/fl knockout striatum (d-f; t(4) = 24.504, P = 0.000016), as assayed by immunofluorescence staining and Western blotting. Scale bars, 100 μm. Error bars represent s.e.m. ***P < 0.001. Images and data represent three mice per genotype. (g) Corticostriatal terminals marked by VGluT1 (green) form putative synapses with PSD95 (red) in P8 striatum. Inset in the middle panel shows adjacent presynaptic VGluT1-positive (green, arrows) and postsynaptic PSD95-positive (red, arrowhead) puncta at high magnification. Scale bars, 10 μm. Images represent two repeats. (h-j) Foxp2 is predominantly expressed by layer VI neurons, whereas Mef2C is expressed throughout the primary motor cortex at P0 (h), P8 (i) and P14 (j). Scale bars, 100 μm. Images represent two repeats.

Supplementary Figure 2 GluR1 immunostaining in the striatum of Foxp2−/−, Foxp2H/H and Nestin-cre;Mef2cfl/fl mice.

(a-c) Double immunostaining of GluR1 and VGluT1 shows that VGluT1-positive striosomes contain high levels of GluR1 expression in the striatum. GluR1 immunofluorescence intensity is decreased in striosomes (t(4) = 4.228, P = 0.013) and matrix (t(4) = 3.535, P = 0.024), as well as entire striatum (t(4) = 4.298, P = 0.013), of P7 Foxp2−/− mice (b), compared to wildtype mice (a). (d-f) By contrast, GluR1 immunofluorescence intensity is increased in striosomes (t(4) = −7.426, P = 0.002) and matrix (t(4) = −4.343, P = 0.012), and in entire striatum (t(4) = −6.013, P = 0.004), of P8 Foxp2H/H mice (e), relative to wildtype mice (d). (g-i) GluR1 immunofluorescence intensity is increased in the matrix (t(4) = −4.201, P = 0.014) and in the entire striatum (t(4) = −3.240, P = 0.032) of P8 Nestin- cre;Mef2 cfl/fl mice (h), relative to control Nestin- cre;Mef2 c+/+ mice (g). *P < 0.05, **P < 0.01. Error bars represent s.e.m. Scale bars, 200 μm. Images and data represent three mice per genotype.

Supplementary Figure 3 Normal striatal compartmentation and a lack of cell death in Foxp2 and Mef2c mutant mice.

(a- h) MOR1 and CalDAG-GEFI immunostaining show that the macroscopic patterns and the regions of MOR1-positive striosomes (a,c,e,g) and CalDAG-GEFI-enriched matrix (b,d,f,h) are not altered in the striatum of P7 Foxp2−/− mice (a,b), P8 Foxp2H/H mice (c,d), P8 Nestin- cre;Mef2 cfl/fl mice (e,f) or P8 Dlx5/6- cre;Foxp2+/−;Mef2 cfl/+ mice (g,h), relative to control mice (Student's t test, a, for rostral, t(4) = 0.257, P = 0.810; for middle, t(4) = -0.334, P = 0.755; for caudal, t(4) = −0.654, P = 0.549; b, for rostral, t(4) = −0.505, P = 0.640; for middle, t(4) = 0.330, P = 0.758; for caudal, t(4) = 1.392, P = 0.236; c, for rostral, t(4) = −0.035, P = 0.974; for middle, t(4) = −0.816, P = 0.460; for caudal, t(4) = −0.037, P = 0.972; d, for rostral, t(4) = 0.350, P = 0.744; for middle, t(4) = −0.037, P = 0.972; for caudal, t(4) = −0.236, P = 0.825; e, for rostral, t(4) = −0.035, P = 0.283; for middle, t(4) = −0.816, P = 0.341; for caudal, t(4) = −0.037, P = 0.850; f, for rostral, t(4) = 0.210, P = 0.844; for middle, t(4) = 1.029, P = 0.362; for caudal, t(4) = 1.655, P = 0.173; g, for rostral, t(4) = −1.501, P = 0.208; for middle, t(4) = 0.873, P = 0.432; for caudal, t(4) = −0.786, P = 0.476; h, for rostral, t(4) = 0.157, P = 0.883; for middle, t(4) = 0.374, P = 0.728; for caudal, t(4) = −1.669, P = 0.171). Error bars represent s.e.m. Scale bars, 200 μm. (i-q) The number of cells positive for activated caspase 3 (AC3) are not altered in the striatum of P0 (i-k, t(4) = 0.390, P = 0.716) and P8 (l-n, t(4) = −0.940, P = 0.400) Foxp2−/− knockout mice or in the striatum of E18.5 Nestin- cre;Mef2 cfl/fl knockout mice (o-q, t(4) = −0.707, P = 0.519). CTX: cortex; ST: striatum; Sep: septum; cc: corpus callosum. (r) TUNEL signals (left) are not detected in the striatum of P8 Nestin- cre;Mef2 c+/+ (middle) or in the striatum of P8 Nestin- cre;Mef2 cfl/fl (right) mice. Arrows indicate DNase I-treated cells as positive control. (s,t) The density of Foxp2-positive cells in Nestin- cre;Mef2 cfl/fl knockout striatum is similar to that in Nestin- cre;Mef2 c+l+ control striatum (t(4) = −1.800, P = 0.146). (u) Consistently, the protein level of Foxp2 is not altered in P8 Nestin- cre;Mef2 cfl/fl knockout striatum, compared to Nestin- cre;Mef2 c+l+ control striatum (t(4) = 0.569, P = 0.627). Scale bars, 200 μm. Images and data represent three mice per genotype.

Supplementary Figure 4 Alterations of dendritic spines of SPNs in the dorsomedial striatum of Foxp2 and Mef2c mutant mice.

Dendritic spines of SPNs observed in Golgi-stained sections through the dorsomedial striatum are decreased in the Foxp2−/− mice (a, for stubby, F(2, 87) = 1.309, P = 0.275; for thin/filopodia, F(2, 87) = 15.251, P = 0.000002; for mushroom, F(2, 87) = 10.333, P = 0.000094; for branched, F(2, 87) = 6.699, P = 0.002; for atypical, F(2, 87) = 1.882, P = 0.158; for sum, F(2, 87) = 28.470, P = 0.000000), but are increased in Foxp2H/H mice (b, for stubby, t(29) = −0.421, P = 0.677; for thin/filopodia, t(29) = −2.134, P = 0.041; for mushroom, t(29) = −1.670, P = 0.106; for branched, t(29) = −0.867, P = 0.393; for multiple branched, t(29) = −1.795, P = 0.083; for atypical, t(29) = 0.226, P = 0.823; for sum, t(29) = −4.016, P = 0.000383), in Nestin- cre;Mef2 cfll+ and Nestin- cre;Mef2 cfl/fl mice (c, for stubby, F(2, 110) = 61.760, P = 0.000000; for thin/filopodia, F(2, 110) = 12.941, P = 0.000009; for mushroom, F(2, 110) = 16.513, P = 0.000001; for branched, F(2, 110) = 14.529, P = 0.000003; for multiple branched, F(2, 110) = 9.961, P = 0.000106; for atypical, F(2, 110) = 9.982, P = 0.000105; for sum, F(2, 110) = 86.254, P = 0.000000) and in Dlx5/6- cre;Foxp2+/−;Mef2 cfl/+ mice (d, for stubby, F(2, 89) = 3.391, P = 0.038; for thin/filopodia, F(2, 89) = 22.339, P = 0.000000; for mushroom, F(2, 89) = 8.687, P = 0.000366; for branched, F(2, 89) = 10.016, P = 0.000128; for multiple branched, F(2, 89) = 2.433, P = 0.094; for atypical, F(2, 89) = 3.759, P = 0.027; for sum, F(2, 89) = 57.917, P = 0.000000. Atyp: atypical. One-way ANOVA is used in a, c, and d. Student’s t test is used in b. *P < 0.05, **P < 0.01; ***P < 0.001. Error bars represent s.e.m. Scale bars, 2.5 μm. Data represent at least 30 cells from three mice per genotype.

Supplementary Figure 5 Validation of HSV-Cre-GFP virus-mediated deletion of humanized Foxp2 in striatal neurons of Foxp2H/H mice and deletion of Mef2c in striatal neurons of Mef2cfl/fl mice.

(a) Intrastriatal injections of HSV-Cre-GFP viruses were made in P2 Mef2 cfl/fl mice. The boxed region in the left panel, covering the needle track, is shown at high magnification in the right panel. Many GFP-positive cells (arrows in the right panel) are distributed in the region around the needle track (arrowheads in the left panel) in the P8 striatum (ST). CTX, cortex; Sep, Septum. (b,c) GFP-positive cells co-expressing Mef2C (arrows) are found in control wildtype mice (b), but not in P8 HSV-Cre-GFP;Mef2 cfl/fl mice (arrows, c). The sections are double-immunostained for Mef2C and the striatal matrix marker CalDAG-GEFI (left panels) to identify GFP-positive cells in Mef2C-enriched matrix regions. Images represent six repeats. (d,e) Double-immunostaining show co-localization of Foxp2 (red) and GFP in the striatum of P8 HSV-Cre-GFP;Foxp2+/+ wildtype mice (arrows in d), but not in the striatum of HSV-Cre-GFP;Foxp2H/H mice (arrows in e). Images represent four repeats. Scale bars are 200 μm in a, 20 μm in b and d.

Supplementary Figure 6 Mef2C is not appreciably altered in the neocortex of Foxp2 knockout mice, and synaptic markers and Mef2C expression are inversely correlated in the striatum of Foxp2−/− and Foxp2H/H mice during postnatal development.

(a-c) In Foxp2-rich layer VI of primary motor cortex (M1) and primary somatosensory cortex (S1), the density of Mef2C-positive cells is not changed significantly in Foxp2−/− knockout mice compared to Foxp2+/+ wildtype control at P8 (Student’s t test, for M1, t(4) = −2.102, P = 0.103; for S1, t(4) =−2.063, P = 0.108). (d) Western blotting shows no change in the level of Mef2C protein in the neocortex of Foxp2−/− knockout mice (Student’s t test, t(4) = 1.243, P = 0.340). (e-g) In Foxp2−/− mice, the levels of Mef2C and synaptic markers GluR1, PSD95 and VGluT1 are not changed at P4 (e, for GluR1, t(4) = 1.859, P = 0.204; for PSD-95, t(4) = −2.138, P = 0.166; for VGluT1, t(4) = −2.086, P = 0.105; for Mef2C, t(4) = 0.167, P = 0.883). By P8, GluR1, PSD95 and VGluT1 are decreased, but Mef2C is increased (f, for GluR1, t(4) = 3.169, P = 0.034; for PSD-95, t(4) = 3.055, P = 0.038; for VGluT1, t(4) = 4.114, P = 0.015; for Mef2C, t(4) = −2.919, P = 0.043). This pattern of protein expression is maintained at P12 (g, for GluR1, t(4) = 11.890, P = 0.000287; for PSD-95, t(4) = 2.823, P = 0.048; for VGluT1, t(4) = 3.226, P = 0.032; for Mef2C, t(4) = −2.789, P = 0.049). (h-j) In Foxp2 H/H mice, synaptic markers GluR1 and VGluT1 are increased, but Mef2C is decreased at P4 (h, for GluR1, t(4) = −2.952, P = 0.042; for PSD-95, t(4) = 2.020, P = 0.113; for VGluT1, t(4) = −4.518, P = 0.011; for Mef2C, t(4) = 5.838, P = 0.004). By P8, Mef2C is also decreased, but VGluT1, PSD95 and GluR1 are increased (i, for GluR1, t(4) = −6.268, P = 0.003; for PSD-95, t(4) = −2.826, P = 0.048; for VGluT1, t(4) = −5.797, P = 0.004; for Mef2C, t(4) = 3.392, P = 0.027). This pattern of altered protein expression persisted through P21 (j, or GluR1, t(4) = −7.169, P = 0.002; for PSD-95, t(4) = −7.396, P = 0.002; for VGluT1, t(4) = −2.958, P = 0.042; for Mef2C, t(4) = 10.873, P = 0.000406). *P < 0.05, **P < 0.01; ***P < 0.001. Error bars represent s.e.m. Images and data represent three mice per genotype.

Supplementary Figure 7 FOXP2 represses human MEF2C reporter gene activity.

(a) Schematic drawing of 5' flanking region (−872 to 261 bp, transcription initiation site: +1) of human MEF2C gene containing putative hFOXP2 binding sites. (b) Co-transfection of the human MEF2C-c-fos-Luc reporter gene together with hFOXP2, but not co-transfection with hFOXP2R553H mutant (mt-hFOXP2), suppresses Luc reporter gene activity in SH-SY5Y cells. Data represent four repeats. Student’s t test, Mock vs. hFoxp2, t(6) = 8.032, P = 0.003; hFoxp2 vs. mtFoxp2, t(6) = −6.723, P = 0.006. *P < 0.05, **P < 0.01. Error bars represent s.e.m.

Supplementary Figure 8 Analyses of multiple USVs in different Foxp2 mutant mice.

(a) Reduction of multiple USVs in Foxp2+/− heterozygous mice at P8. Compared to Dlx5/6- cre;Foxp2+/+;Mef2 c+/+ mice (white, Foxp2+/+ wildtype), the Dlx5/6- cre;Foxp2+/−;Mef2 c+/+ mice (blue, Foxp2+/− heterozygotes) are defective in multiple USV characteristics, including the number of calls (events; Student’s t test, t(20) = 3.902, P = 0.004), duration of each call (t(20) = 3.870, P = 0.001), number of elements in each call (t(20) = 4.965, P = 0.0001), proportion of calls with frequency jump (t(20) = 4.494, P = 0.001), peak frequency at end of calls (t(20) = 2.765, P = 0.012), peak amplitude at end of calls (t(20) = 3.615, P = 0.002) and maximum peak amplitude (t(20) = 3.782, P = 0.001). Peak frequency at start (t(20) = 0.316, P = 0.756), peak amplitude at start (t(20) = 1.364, P = 0.188) and maximum peak frequency (t(20) = 0.790, P = 0.443) are not affected. (b) Some USV characteristics are not affected by inactivation of one allele of Mef2 cfl/+ in Dlx5/6- cre;Foxp2+/− heterozygotes (yellow). The duration of each call (one-way ANOVA followed by Tukey’s HSD post hoc test, F(2, 36) = 7.317, P = 0.002), peak frequency at start (F(2, 36) = 1.590, P = 0.218), peak amplitude at start (F(2, 36) = 0.861, P = 0.431), peak amplitude at end (F(2, 36) = 5.729, P = 0.007), maximum peak frequency (F(2, 36) = 2.705, P = 0.080) and maximum peak amplitude (F(2, 36) = 6.949, P = 0.003) of USV are not statistically altered in Foxp2 heterozygotes mice in which Mef2 c is heterozygous (Dlx5/6- cre;Foxp2+/−;Mef2 cfl/+) relative to USVs in Dlx5/6- cre;Foxp2+/−;Mef2 c+/+ mice in which Mef2 c is wildtype. (c) USV analyses of P8 Dlx5/6- cre;Foxp2+/+;Mef2 cfl/+ (green) and Dlx5/6- cre;Foxp2+/+;Mef2 c+/+ (white) mice. No significant changes in USV features are found between these mice (Student’s t test, for events, t(19) = −0.664, P = 0.515; for duration, t(19) = −0.552, P = 0.587; for elements, t(19) = −1.099, P = 0.286; for frequency jump, t(19) = −0.720, P = 0.480; for peak frequency at start, t(19) = −0.031, P = 0.976; for peak amplitude at start, t(19) = −0.921, P = 0.369; for peak frequency at end, t(19) = −0.968, P = 0.345; for peak amplitude at end, t(19) = −1.263, P = 0.222; for maximum peak frequency, t(19) = −0.747, P = 0.464; for maximum peak amplitude, t(19) = −0.031, P = 0.976). (d) Some USV characteristics are not affected in HSV-Cre-GFP;Foxp2+/−;Mef2 cfl/+ mice. The duration of each call (Student’s t test, t(14) = 0.046, P = 0.964), elements (t(14) = 1.483, P = 0.160), peak frequency at start (t(14) = −2.104, P = 0.054), peak amplitude at start (t(14) = 0.096, P = 0.350), peak frequency at end (t(14) = 0.053, P = 0.959), peak amplitude at end (t(14) = −0.943, P = 0.362), maximum peak frequency (t(14) = −0.663, P = 0.518) and maximum peak amplitude (t(14) = −0.650, P = 0.526) of USVs are not statistically altered in P8 HSV-Cre-GFP;Foxp2+/−;Mef2 cfl/+ mice (red) compared to control HSV-Cre-GFP;Foxp2+/−;Mef2 c+/+ mice (white). Box plots show the median (horizontal line in the box), range between the 25th and 75th percentiles (box), and 1.5 times this interquartile range (T-bars). Outlying values are marked as circles. **P < 0.01; ***P < 0.001. Data represent at least 8 mice per genotype.

Supplementary Figure 9 Schematic drawings summarizing striatal Foxp2–Mef2C interaction in regulating synaptogenesis and vocalization.

(left) VGluT1+ corticostriatal axon terminals preferentially innervate immature striosomal SPNs (Foxp2high/Mef2Clow) but not immature matrix SPNs (Foxp2low/Mef2Chigh) at P0-P8 (top). By P14, corticostriatal axons innervate both striosomal and matrix cells (Foxp2high/Mef2Clow, bottom). (right) Diagram depicting hypothesis that Foxp2 functions as a molecular key to unlock Mef2C-mediated inhibition of synapse formation in SPNs during development. When Foxp2 levels are low, Mef2C is de-repressed to inhibit corticostriatal synaptogenesis (top). When Foxp2 levels are high, Foxp2 represses Mef2C to promote corticostriatal synaptogenesis, which is linked to facilitation of USV production (bottom).

Supplementary Figure 10 Full-length gels and blots.

The original Western blots of the cropped blots shown in Figures 3a-c, 4d,e,h and 6e, and in Supplementary Figures 1c,f, 3u and 6d-j, and the original gel shown in Figure 5b.

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Chen, YC., Kuo, HY., Bornschein, U. et al. Foxp2 controls synaptic wiring of corticostriatal circuits and vocal communication by opposing Mef2c. Nat Neurosci 19, 1513–1522 (2016). https://doi.org/10.1038/nn.4380

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